Antidiuretic hormone (ADH) controls total body water balance by changing tight epithelial cell solute and water permeability. ADH increases the water permeability (Pf) of the apical membranes of cells in the mammalian kidney and anuran urinary bladder by the vesicle-mediated insertion of highly selective water channels. ADH water channels are believed to be composed of proteins based on biophysical data inhibition of ADH Pf by mercurial reagents. Despite their importance, the structures of ADH water channel proteins are not known. In an effort to do so, our laboratory has characterized the membrane proteins of vesicles called aggrephores that store large numbers of ADH water channels in toad urinary bladder. These vesicles are highly water and proton permeable. Proteins of 55 and 53 kilodaltons (kD) are the predominant species in these vesicles and both span the membrane of these vesicles. Both proteins appear exclusively in the apical membrane only after ADH stimulation and both have a large cysteine content. Their derivatization with fluorescein mercuric acetate is associated with an 82% inhibition of vesicle Pf. Rabbit antisera raised against these 55 and 53 kD proteins inhibits ADH elicited Pf by 78%. These data suggest these proteins are ADH water channel components. Recently, we have identified clones from a toad bladder lambdalgt11 library using this antisera and demonstrated that water channels are expressed in Xenopus oocytes after injection of toad bladder poly A+ RNA. We propose to first, characterized the nucleotide sequence and expression of the 55 and 53 kD proteins in toad bladder; second, we will determine if their transcripts are responsible for expression of water channels in oocytes and third, we will examine the functional topology of these water channel proteins within the membrane, test whether functional ADH water channels are composed of multiple subunits and if they are present in other tissues besides the toad bladder.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK038874-07
Application #
2140715
Study Section
General Medicine B Study Section (GMB)
Project Start
1987-07-01
Project End
1996-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
7
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
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